Fast pressure jumps can perturb calcium and magnesium binding to troponin C F29W.
We have used rapid pressure jump and stopped-flow fluorometry to investigate calcium and magnesium binding to F29W chicken skeletal troponin C. Increased pressure perturbed calcium binding to the N-terminal sites in the presence and absence of magnesium and provided an estimate for the volume change upon calcium binding (-12 mL/mol). We observed a biphasic response to a pressure change which was characterized by fast and slow reciprocal relaxation times of the order 1000/s and 100/s. Between pCa 8-5.4 and at troponin C concentrations of 8-28 muM, the slow relaxation times were invariant, indicating that a protein isomerization was rate-limiting. The fast event was only detected over a very narrow pCa range (5.6-5.4). We have devised a model based on a Monod-Wyman-Changeux cooperative mechanism with volume changes of -9 and +6 mL/mol for the calcium binding to the regulatory sites and closed to open protein isomerization steps, respectively. In the absence of magnesium, we discovered that calcium binding to the C-terminal sites could be detected, despite their position distal to the calcium-sensitive tryptophan, with a volume change of +25 mL/mol. We used this novel observation to measure competitive magnesium binding to the C-terminal sites and deduced an affinity in the range 200-300 muM (and a volume change of +35 mL/mol). This affinity is an order of magnitude tighter than equilibrium fluorescence data suggest based on a model of direct competitive binding. Magnesium thus indirectly modulates binding to the N-terminal sites, which may act as a fine-tuning mechanism in vivo.
Pearson
David S
DS
Department of Biosciences, University of Kent, Canterbury CT2 7NJ, UK.
Swartz
Darl R
DR
Geeves
Michael A
MA
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002645:Chickens; D008274:Magnesium / Q000737:chemistry* / Q000378:metabolism; D008956:Models, Chemical; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000378:metabolism; D020125:Mutation, Missense; D011312:Pressure; D011485:Protein Binding / Q000235:genetics; D013050:Spectrometry, Fluorescence / Q000379:methods; D019209:Troponin C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D019209:Troponin C; D008274:Magnesium; D002118:Calcium